miRCURY LNA miRNA Custom Bulk Plate are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

Schematic outline of the miRCURY LNA miRNA PCR System.

A poly(A) tail is added to the mature miRNA template (step 1A). cDNA is synthesized using a Poly(T) primer with a 3’ degenerate anchor and a 5’ universal tag (step 1B). The cDNA template is then amplified using two miRNA-specific and LNA-enhanced forward and reverse primers (step 2A). SYBR Green is used for detection (step 2B).

A 10-fold serial dilution (ranging from 1 x 108 to 10 copies) of cDNA template made from synthetic hsa-miR-181a was used for real-time PCR amplification. The standard curve shows excellent linear correlation (R2 = 0.998) between the decreasing cycle numbers and the logarithm of the miRNA copy number. The assay can detect as few as 10 miRNA copies in the PCR reaction and has a dynamic range of 8 logs. Efficiency calculated from the standard curve = 1.94. The dilution series was performed in a background of MS2 bacteriophage total RNA.

miRCURY LNA miRNA PCR System at a glance.

The miRCURY LNA miRNA PCR System uses one single cDNA synthesis reaction for all amplifications, reducing pipetting and saving time and sample. Two LNA-enhanced miRNA-specific qPCR primers enable highly specific and sensitive amplification and single nucleotide discrimination. The fast and easy workflow takes only 3 hours with minimal hands-on steps.

Specificity test alongside competitor probe-based miRNA qPCR system.

Specificity was assessed using 8 pools each containing at least 80 different synthetic miRNAs. Closely related miRNAs are placed in different pools. This example shows results from the hsa-miR-1 assay. The miRCURY LNA miRNA PCR System performs perfectly with a high signal in pool 1, which contains the hsa-miR-1 synthetic miRNA target, and only a borderline signal in pool 7, which contains the most closely related miRNA (hsa-miR-206). The competitor platform uses ligation-based cDNA synthesis and only one miRNA-specific probe. Competitor T shows a lack of specificity. The highest signal for Competitor T is obtained with pool 1. However, non-specific false-positive signals are obtained from all other pools in the absence of hsa-miR-1 template, within a Cq range that would be interpreted as real signals.

Detection of differential expression of miRNAs in LCM specimens from tissue FFPE sections.

Areas between 8000 and 13000 μm2 of normal tissue, tumor tissue and tumor stroma were isolated by laser dissection from FFPE sections of human colon (A). Total RNA was extracted and miRNA levels were quantified using miRCURY LNA miRNA PCR (B). Normalized data are shown on a log2 scale as relative expression compared to normal tissue. hsa-miR-103 and hsa-let-7a were used as reference genes.

Differences in miRNA expression between serum samples.

Normalized expression levels of eight different miRNAs in two serum samples are shown. Total RNA purified from the equivalent of 8 µl serum was used in the RT reaction. hsa-let-7a and hsa-miR-103 were used as reference genes for normalization.

Specific and sensitive amplification of miRNA.

Amplification curves (A) and melting curves (B) from the hsa-miR-145 miRCURY LNA miRNA PCR Assay performed on a serial dilution of human colon total RNA (100 ng to 1 pg), performed in triplicate. The amplification curves demonstrate sensitive and reproducible detection down to 1 pg of total RNA input with no background signal. The melting curves show only one major Tm peak corresponding to a well-defined melting temperature of the amplicon, demonstrating specific amplification of hsa-miR-145.

Single nucleotide discrimination.

The LNA-enhanced, miRNA-specific primers facilitate the design of miRNA qPCR assays that discriminate between closely related miRNA sequences. The table shows the percent signal obtained from target with a single nucleotide mismatch compared to the signal from the perfectly matched target (set to 100%). Single nucleotide discrimination is possible at various positions in the miRNA sequence.

Performance

The design process

The miRCURY LNA miRNA Custom PCR Assay design tool lets you easily design highly sensitive and specific LNA-enhanced PCR primer sets for any miRNA not available as a predesigned product. The advanced algorithm evaluates approximately 3,000 primer pair designs based on more than 60 different criteria to find LNA optimal primer sets for your miRNA within a few minutes. The tool has been designed for miRNAs but can also be used for other small RNAs 14–27 nucleotides in length.

The design criteria include:

Optimization of melting temperatures by varying the LNA distribution of the primers to ensure high amplification efficiency and specificity.

Calculations and adjustments of self-hybridization and cross-hybridization scores for efficient PCR reactions.

Adjustments to avoid potential primer–dimer formation in the PCR reaction.

Intelligent positioning of LNA bases in the primers based on our vast knowledge of LNA oligonucleotide design.

miRBase searches to identify potential miRNAs with high sequence similarity. This ensures that the primer sets are highly specific for the small RNA for which they were designed.

Unmatched sensitivity

The exceptional sensitivity of miRCURY LNA miRNA PCR Assays is achieved by combining universal reverse transcription with LNA-enhanced and Tm-normalized primers. This combination enables accurate and reliable quantification of individual miRNAs from as little as 1 pg of total RNA input in the initial first-strand cDNA synthesis (see figure Accurate quantitation from down to 1 pg total RNA starting material).

Fully validated and optimized

All wet-lab validated miRCURY LNA miRNA PCR Assays have been optimized to be as sensitive as possible. Over 80% of assays detect at least 5 miRNA copies in the PCR reaction (see figure Serial dilution of hsa-miR-181a). The primer sets have also been validated for specific amplification of the target and for minimal background signal (see figure Specific and sensitive amplification of miRNA).

Superior discrimination

The incorporation of LNA in both the forward and reverse PCR amplification primers makes it possible to design assays that can distinguish between miRNA sequences that differ by only one nucleotide (see figure Single-nucleotide discrimination). In addition, the assays can discriminate between mature miRNA sequences and precursor miRNA.

Fast, easy and reproducible

The easy-to-follow, 3-hour protocol saves you both time and effort in the laboratory. By using the same RT reaction as the template in all subsequent PCR reactions, the procedure is greatly simplified compared with systems that require miRNA-specific first-strand synthesis. The number of pipetting steps is reduced to a minimum, and technical variation is minimized. This makes it possible to achieve extremely high reproducibility from day-to-day and even site-to-site.

miRCURY LNA miRNA PCR Assays have been optimized for use with the miRCURY LNA RT Kit and the miRCURY LNA SYBR Green PCR Kit. Use of other reagents will affect the quality of the results.

Principle

A unique system for miRNA profiling

miRCURY LNA miRNA PCR Assays offer the best combination of performance and ease-of-use available on the miRNA real-time PCR market by combining universal RT with LNA PCR amplification (see figure Schematic outline of the miRCURY LNA miRNA PCR System). Universal RT makes it possible to use one first-strand cDNA synthesis reaction as the template for multiple miRNA real-time PCR assays. This saves precious samples, reduces technical variation and saves time in the laboratory. Plus, both the forward and reverse PCR amplification primers are miRNA specific and optimized with LNA. This provides 1) exceptional sensitivity and extremely low background, enabling accurate quantitation of very low miRNA levels and 2) highly specific assays that allow discrimination between closely related miRNA sequences.

Coverage

Over 20,000 assays are available covering all organisms in miRBase 20. Over 1,400 assays are fully wet-lab validated for sensitivity, specificity, efficiency and background on both synthetic as well as different biological samples. The remaining assays are in silico-validated using a comprehensive design algorithm that ensures high-quality, species-specific, LNA-enhanced assays with optimal sensitivity and specificity within each organism. This means that several different assays may target the same sequence. Ultimately, the assay for each species is selected based on the genetic background of the organism. If you are working with novel miRNAs, such as from an NGS experiment, custom-designed LNA miRNA primer sets for any miRNA are also available.

Procedure

The miRCURY LNA miRNA PCR Assay system is an miRNA-specific, LNA-based system designed for sensitive and accurate detection of miRNA by quantitative real-time PCR using SYBR Green. The first step of the procedure is universal reverse
transcription, followed by real-time PCR amplification with LNA-enhanced primers (see figure miRCURY LNA miRNA PCR System at a glance).

Applications

miRCURY LNA miRNA PCR Assays are used as part of the miRCURY LNA miRNA PCR System for: